Nucleic acid amplification method

Abstract

An object to be achieved by the present invention is to provide a nucleic acid amplification method by which a nucleic acid can be amplified substantially isothermally using oligonucleotide primers and DNA polymerase capable of strand displacement. The present invention provides a nucleic acid amplification method which comprises performing substantially isothermal incubation of a reaction solution containing at least one type of deoxynucleotide triphosphate, at least one type of DNA polymerase having strand displacement activity, a divalent cation, at least 0.01% or more surfactant, at least two types of oligonucleotide primer, and the nucleic acid fragment as a template so as to perform a polymerase reaction that initiates from the 3′ end of the primer and thus amplifying the nucleic acid fragment.

Description

TECHNICAL FIELD[0001]The present invention relates to a nucleic acid amplification method. More specifically, the present invention relates to a nucleic acid amplification method that comprises performing a polymerase reaction through substantially isothermal incubation of a reaction solution using DNA polymerase capable of strand displacement.BACKGROUND ART[0002]In molecular biological research, nucleic acid amplification is generally performed by an enzymatic method using DNA polymerase. Polymerase chain reaction (PCR) is broadly known as a nucleic acid amplification method. For amplification of a target nucleic acid sequence, the PCR method comprises the three steps of: denaturing (denaturation step) double-stranded DNA as a template into single-stranded DNAs; annealing (annealing step) primers to the single-stranded DNAs; and elongating (elongation step) complementary strands using the primers as origins. According to a general PCR method, the denaturation step, the annealing st...

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Benefits of technology

[0008]An object to be achieved by the present invention is to provide a nucleic acid amplification method by which a nucleic acid can be amplified substantially isothermally using oligonucleotide primers and DNA polymerase capable of strand displacement. Furthermore, an object to be achieved by the present invention is to provide a nucleic acid amplification method by which a target nucleic acid sequence can be amplified in a short time and a target nucleic acid sequence can be specifically amplified. Furthermore, an object to be achieved by the present invention is to provide a nucleic acid amplification method with a simpler primer design.

[0009]As a result of intensive studies to achieve the above objects, the present inventors have discovered that a nucleic acid fragment can be efficiently amplified within a short time when a polymerase reaction that is initiated from the 3′ end of a primer is performed through substantially isothermal incubation of a reaction solution containing deoxynucleotide triphosphate, DNA polymerase capable of strand displacement, a divalent cation, a surfactant, oligonucleotide primers, and a nucleic acid fragment as a template. Thus, the present inventors have completed the present invention. The oligonucleotide primer used in the present invention is characterized in that it has no complicated structures such as those used in the conventional isothermal amplification methods. For example, it is not necessary that the oligonucleotide primer has a structure which forms a chimera structure which is used in the ICAN method or a loop structure which is used in the LAMP method.

[0037]According to the present invention, a target nucleic acid sequence can be amplified substantially isothermally. Moreover, according to the present invention, a target nucleic acid sequence can be amplified specifically within a short time.

Problems solved by technology

However, implementation of nucleic acid amplification reactions at three different types of temperature is problematic in that temperature control is complicated and time loss increases in proportion to the number of cycles.

However, the use of exonuclease in addition to polymerase is required, and thus the method is expensive and the design of primers should be improved.

However, the method requires the use of at least four types of primer that recognize six specific sites, so that the design of primers is very difficult.

However, this method also requires the use of special primers that are chimeric primers and thus the design of such primers is very difficult.

However, the method disclosed in JP Patent Publication (Kohyo) No. 11-509406 A is problematic in that it requires a relatively long reaction time, for example.

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